Project Summary: My long-range goal is to become an independent investigator seeking novel ways to diagnose and treat congenital heart disease, heterotaxy and other human birth defects. Birth defects are the major cause of infant death in the US and Europe, but very little is known about their causes. My research will focus on the pathophysiology underlying an important type of birth defect--namely, heterotaxy, in which numerous organs (e.g., heart, lungs, liver) are abnormally arranged in the torso due to defects in left-right (LR) patterning during embryogenesis. More than 90% of infants with heterotaxy have severe congenital heart disease and display low survival rates (as low as 30%) despite surgical intervention. In a growing number of cases, genetic studies have traced heterotaxy to defects in the structure and function of cilia, hair-like organelles that are found on cells of nearly all developing organs. Using zebrafish as a model organism, I have obtained preliminary evidence that cilia act as ?antennae? to sense and translate environmental cues, such as extracellular fluid flow, into calcium signals that sculpt the heart during early embryogenesis. These results suggest that cilia serve a mechanotransduction function during development; however, little is known about the molecular machinery that executes this process in the cilium. Strikingly, by targeting calcium reporters into the cilium, I have detected calcium signaling within the cilium that may contribute to this function. To gain greater mechanistic understanding into ciliary sensation and intraciliary calcium signaling, I propose three Aims that will provide an opportunity to train and implement specific goals. In Aim 1, I will investigate how polycystin-2 (Pkd2), a ciliary cation channel, initiates intraciliary calcium signaling during the development of the left-right axis. I will specifically look at how Pkd2 interacts with a binding partner, Pkd1l1, to sense fluid flow and release calcium in the cilium. In Aim 2, I will examine how Inversin (Invs), a calcium-sensitive protein that localizes to the base of the cilium, functions as a transducer by relaying intraciliary calcium signals into the cell body. By studying mice that lack normal Inversin protein, which results in 100% organ reversal, I will gain understanding into how transduction of ciliary calcium is critical for left-right development. In these 2 Aims, I will gain training in polycystin channels, mouse genetics and calcium physiology. In Aim 3, I will investigate if the cilium is a calcium signaling compartment that responds to mechanical force during left-right development. In this first part of this Aim, I will be trained in biophysical manipulation techniques, such as optical tweezers, to apply controlled force in order to mechanically bend the cilium. I will then utilize these techniques in the R00 phase to characterize the mechanosensitive machinery of the cilium and to address whether ciliary mechanotransduction and intraciliary calcium signaling are instructive for left-right patterning. Completion of these Aims will elucidate the pathophysiology underlying cilia-related birth defects and may guide rational design of novel therapeutic tools for the diagnosis and possibly even treatment of cilia-associated birth defects.